The O-glycosylation of mucin-type molecules is responsible for their unique physicochemical properties. Despite the fact that O- glycosylation is ubiquitous, little is known about the way it is regulated. We are studying the initiation of glycosylation by UDP- N-acetylgalactosamine:peptide N-acetylgalactosamine transferase. We have formed an extensive database of known glycosylated sites and their flanking sequences. Computer analysis of the amino acid sequences surrounding glycosylated site does not reveal a strict consensus, but suggests that sequence may influence the initiation of O-glycosylation, particularly at positions -1 and +3, relative to the glycosylated site. A series of test peptides was made to experimentally verify this hypothesis. It was found that single amino acid changes at the 'sensitive' positions near a threonine residue could dramatically alter its ability to be glycosylated. Furthermore, circular dichroism spectroscopy showed that all the substrates that were glycosylated had random-type structure. A set of 50 peptides has been synthesized to extend the investigation of flanking sequences. Serine-containing substrates glycosylate poorly compared to those containing threonine. GalNAc transferase was partially purified from a number of rat, human and bovine tissue. Assays of serine- containing peptides reveals that their inability to be glycosylated in vitro is not due to flanking sequence alone.. We have suggested that a separate GalNAc transferase is responsible for glycosylating serine, and are currently attempting to isolate this activity. It seems possible that a family of GalNAc transferases exists, having multiple overlapping sequence specificities. This would explain the absence of a single consensus sequence for all glycosylated sites. Future studies, involving the design and production of recombinant mucin-like species (eg for replacement therapy in salivary hypofunction) will have to take into account both substrate sequence requirements and enzyme activities available to successfully facilitate their glycosylation.